Boophilus microplus Anticoagulant Protein: An Antithrombin Inhibitor Isolated from the Cattle Tick Saliva Fabiana Horn,* , † ,1 Patrı ´cia Coutinho dos Santos,* and Carlos Termignoni* , ‡ *Centro de Biotecnologia, †Departamento de Biofı ´sica, and ‡Departamento de Bioquı ´mica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brasil Received May 1, 2000, and in revised form August 23, 2000 An anticoagulant was isolated from saliva of the cat- tle tick Boophilus microplus. Crude saliva prolonged both recalcification time and prothrombin time in as- says with bovine plasma. It also inhibited thrombin, but not fXa, amidolytic activity. We purified the anti- thrombin activity by a combination of gel filtration, anion exchange, and affinity chromatography. The pu- rified inhibitor has a molecular weight of 60,000 Da, determined by SDS–PAGE. The anticoagulant IC 50 var- ied from 100 nM to 1.1 M, depending on the thrombin concentration and substrate used (fibrinogen or plate- let receptor). The excess of inhibitor in relation to thrombin indicates that it is not a tight-binding inhib- itor. Chromogenic assays using a panel of five serine- proteinases suggest that the inhibitor is specific against thrombin. © 2000 Academic Press Key Words: tick anticoagulant; antithrombin; Boophilus; tick saliva; thrombin inhibitor. Antihemostatic substances from the saliva of blood- sucking animals are crucial to facilitate hematophagy. Analyses of saliva from such organisms reveal the presence of inhibitors targeting one or more coagula- tion factors that lead to fibrin clot formation (1). Anti- coagulants’ most usual targets are factor Xa and thrombin, which are common to both pathways of co- agulation. Thrombin plays a central role in hemostasis, not only for converting fibrinogen into fibrin but also for regulating its own production by the activation of other coagulation factors (fV, fVIII, fXI, and protein C). Furthermore, thrombin is a potent inducer of platelet aggregation. This enzyme is unique in its specificity: besides its active site, thrombin contains at least one region in its surface that mediates the binding to fibrinogen, platelet receptor and thrombomodulin, greatly enhancing its substrate selectivity (2). Thrombin inhibitors evolved independently in a va- riety of organisms (3–5). In ticks, antithrombin sub- stances have been found in Ornithodorous species (or- nithodorin (6), savignin (7)), Amblyomma americanum (8), and Ixodes ricinus (9). We investigated salivary anticoagulants of the cattle tick, Boophilus microplus. In this paper, we present the identification of a 60-kDa inhibitor from B. microplus, specific for thrombin. MATERIALS AND METHODS Materials Thrombin-Sepharose CL-4B affinity resin was prepared by cou- pling thrombin (Sigma-Aldrich) previously treated with H-D-Phe- Pro-Arg-chloromethylketone (PPACK, 2 from Bachem, USA) (10) to CNBr-activated Sepharose as described by the manufacturer (Phar- macia). Factor Xa, trypsin, chymotrypsin, activated protein C, and plasmin were from Sigma-Aldrich. Thrombin used for the assays was prepared according to the method described in Ding and Xu (11). Chromogenic substrates S-2238, S-2222, S-2390, and S-2366 were purchased from Chromogenix (Sweden); benzoyl-Phe-Val-Arg-p-ni- troanilide, benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, benzoyl-Arg-p-ni- troanilide, and succinyl-Ala-Ala-Pro-p-nitro-anilide were purchased from Sigma-Aldrich. Harvest of Saliva Female ticks that had spontaneously fallen from the bovine were induced to salivate with pilocarpine. Ticks were maintained in a wet chamber and saliva was collected for a period of 2 h following injec- tion of 5 l of a 2% solution of pilocarpine. On average, yields were 1 l saliva per tick. Saliva was stored at -20°C until use. 1 To whom correspondence should be addressed at Centro de Bio- tecnologia, UFRGS, P.O. Box 15005, CEP 91501-970, Porto Alegre, RS, Brasil. Fax: -55 51 3191079. E-mail: fhorn@dna.cbiot.ufrgs.br. 2 Abbreviations used: PPACK, H-D-Phe-Pro-Arg-chloromethylke- tone; BSA, bovine serum albumin; Pip, L-pipecolyl; SDS–PAGE, so- dium dodecyl sulfate–polyacrylamide gel electrophoresis. 68 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 384, No. 1, December 1, pp. 68 –73, 2000 doi:10.1006/abbi.2000.2076, available online at http://www.idealibrary.com on